A kind of biological fermentation preparation method of quercetin

Abstract

The invention discloses a biological fermentation preparation method of quercetin, which relates to the technical field of bioengineering. Rutin or rutin-rich sophora japonica is used as the main raw material, and the mycelium of Coprinus comatus containing quercetin is obtained by liquid shaking bottle culture of Coprinus comatus, liquid seed tank expansion culture and liquid fermentation to convert rutin. The mycelium is extracted, concentrated and recrystallized from acetone to obtain quercetin. The purity of the quercetin produced by the method is ≥98%, and the quercetin can be used to produce medicines, health products, functional foods, food and beverages, and feed.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a method for preparing quercetin by utilizing Coprinus comatus strains and adopting liquid fermentation technology. Background Art

[0002] Malignant tumors seriously endanger the health and life of the Chinese people, and their incidence and mortality rates are gradually increasing. How to control the occurrence and development of lung cancer has become an important bottleneck in meeting people's needs for a better spiritual life.

[0003] Quercetin is a polyhydroxyflavonoid compound widely found in nature, with a molecular formula of C 15 H 10 O7. Quercetin has multiple biological activities such as anti-inflammatory, antioxidant, antibacterial, anticancer and antiplatelet aggregation. At the same time, the toxic side effects of quercetin are very small, which can avoid the teratogenic, carcinogenic and lethal effects of common drugs on the body, so it has good clinical value and application value. Many studies have shown that quercetin not only has the effect of treating lung cancer. Quercetin has an antioxidant effect, so it can remove free oxygen radicals from cells, reduce the damage of free radicals to the biological membrane in the body, and inhibit the increase of permeability of capillaries in the abdominal cavity when the body is inflamed, thereby reducing the loss of water and electrolytes in the intestine, thereby achieving the purpose of antidiarrhea. In addition, studies have shown that quercetin has the effect of reducing the body's insulin resistance, which shows that quercetin has a significant hypoglycemic effect. In short, the biological activity of quercetin is related to the number and position of its phenolic hydroxyl groups, and the position of the phenolic hydroxyl group plays a vital role in its physiological function. Therefore, quercetin can be used in the production of medicines, health products, functional foods, food and beverages, feed, daily chemical products, etc. The market demand is huge and is gradually increasing.

[0004] Quercetin is widely present in the stems, leaves, flowers, fruits and seeds of plants, such as buckwheat stalks / leaves, sea buckthorn, onions, asparagus, green peppers, lettuce, radishes, potato leaves, tomato leaves, pea leaves, broad bean leaves, apples, mangoes, Sophora japonica, Panax notoginseng, Ginkgo biloba, elderberry, etc., all of which contain quercetin, among which buckwheat stalks / leaves, pea leaves and broad bean leaves have high quercetin content. Due to its widespread presence in plants, quercetin can be prepared by plant separation and extraction methods, but quercetin has a low content in plants, and its separation and extraction difficulty is high, and industrial production generally does not use this production process. In addition, quercetin can also be prepared by acid hydrolysis and enzyme conversion. The acid hydrolysis method is to first extract rutin from plants such as Sophora japonica, and then make quercetin through an acid hydrolysis process; the enzyme conversion method is to use plant extract rutin as raw material, and use enzymes synthesized by biological fermentation to hydrolyze rutin to prepare quercetin. This method can be used for large-scale production of quercetin, and the product is highly safe and has greater development prospects in the future. Summary of the invention

[0005] In view of the high incidence of cancer, especially lung cancer, quercetin has a significant effect on controlling cancer. Although quercetin has a wide range of sources, its content in each plant is low. The purpose of the present invention is to provide a method for obtaining mycelium containing high content of quercetin by liquid fermentation for preparing quercetin, and obtaining quercetin from Coprinus comatus mycelium by ethanol extraction, concentration and acetone recrystallization, wherein the quercetin content is ≥98%.

[0006] After in-depth research, the inventors have found a method of using glycerol, xylose and urea as main raw materials, and obtaining a fermentation liquid containing quercetin through liquid shake flask culture of Coprinus comatus mycelium and liquid seed tank expansion culture. The fermentation liquid is concentrated, extracted with ethanol, extracted with water-saturated n-butanol, and recrystallized to obtain quercetin.

[0007] The present application provides a method for preparing quercetin by biological fermentation, comprising the following steps:

[0008] Step 1, inoculating the Coprinus comatus strain into a potato glucose medium to obtain a Coprinus comatus slant strain;

[0009] Step 2, inoculating the Coprinus comatus slant strain obtained in step 1 into a shake flask culture medium, and culturing to obtain a shake flask strain;

[0010] Step 3, inoculating the shake flask strain obtained in step 2 into the seed tank culture medium at a volume ratio of 5-20%, and cultivating to obtain the seed tank strain;

[0011] Step 4, aseptically transferring the seed tank strain obtained in step 3 into a fermentation medium at an inoculation rate of 5-20% by volume, and culturing to obtain a fermentation liquid containing quercetin;

[0012] Step 5, separating and purifying the fermentation broth obtained in step 4 to obtain pure quercetin;

[0013] The shake flask culture medium, seed tank culture medium and fermentation culture medium contain corn flour, bran, glucose, fructose, glycerol, acid-hydrolyzed soybean protein, KH2PO4, MgSO4, ZnSO4, VB2 and VC.

[0014] Furthermore, the seed tank culture medium contains Sophora japonica bud or rutin, and the fermentation culture medium contains Sophora japonica bud or rutin and urea.

[0015] Further, the concentration of corn flour in the shake flask medium and the seed tank medium is 5-15 g / L, the concentration of bran is 5-25 g / L, the concentration of glucose is 5-25 g / L, the concentration of fructose is 1-20 g / L, the concentration of glycerol is 2-20 g / L, the concentration of acid-hydrolyzed soy protein is 0.5-2 g / L, the concentration of KH2PO4 is 1-2.4 g / L, the concentration of MgSO4 is 0.5-2 g / L, the concentration of ZnSO4 is 0.01-0.05 g / L, the concentration of VB2 is 0.01-0.04 g / L, and the concentration of VC is 0.01-0.06 g / L; the concentration of corn flour in the fermentation medium is 5-30 g / L, the concentration of bran is 5-55 g / L, the concentration of glucose is 10-50 g / L, the concentration of fructose is 1-40 g / L, and the concentration of glycerol is 5-45 g / L, urea concentration is 1-4g / L, acid hydrolyzed soy protein concentration is 1-4g / L, KH2PO4 concentration is 1-2.4g / L, MgSO4 concentration is 0.5-2g / L, ZnSO4 concentration is 0.01-0.05 g / L, VB2 concentration is 0.01-0.04 g / L, and VC concentration is 0.01-0.06 g / L.

[0016] Furthermore, the corn flour concentration in the shake flask medium and the seed tank medium is 10-15 g / L, the bran concentration is 10-25 g / L, the glucose concentration is 15-25 g / L, and the fructose concentration is 11-20 g / L; the corn flour concentration in the fermentation medium is 17-30 g / L, the bran concentration is 30-55 g / L, the glucose concentration is 30-50 g / L, and the fructose concentration is 20-40 g / L.

[0017] Furthermore, the culture conditions of step 2 are 100-200 rpm, 20-32° C., and culture for 3-7 days.

[0018] Furthermore, the culture conditions of step 2 are 100-200 rpm, 20-26° C., and culture for 5-7 days; or 100-200 rpm, 26-32° C., and culture for 3-5 days.

[0019] Furthermore, the culture conditions of step 3 are 20-32° C., ventilation volume 0.1-1.2 / min, rotation speed 60-150 rpm for 2-4 days.

[0020] Furthermore, the culture conditions of step 3 are 24-32° C., ventilation volume 0.4-1.2 / min, rotation speed 60-120 rpm for 2-4 days.

[0021] Furthermore, the culture conditions of step 4 are 20-32° C., aeration 0.1-1.2 / min, rotation speed 60-150 rpm for 24-144 hours.

[0022] Furthermore, the culture conditions of step 4 are 24-32° C., aeration 0.4-1.2 / min, rotation speed 60-150 rpm for 68-144 hours.

[0023] Furthermore, the separation and purification in step 5 further comprises the following steps:

[0024] Step a, centrifuging the fermentation liquid and drying the residue to obtain Coprinus comatus mycelium containing quercetin;

[0025] Step b, extracting the mycelium of Coprinus comatus by reflux extraction with anhydrous ethanol, concentrating the extract and mixing it with deionized water, precipitating, filtering, washing the filter residue with deionized water, and drying;

[0026] Step c: dissolving the dried product with acetone and crystallizing it to obtain pure quercetin.

[0027] Furthermore, in the above step b, the mycelium of Coprinus comatus is mixed with 30 times the volume of anhydrous ethanol, refluxed and extracted at 85° C. for 4 hours, and the extract is concentrated to 1 / 5 of the original volume and mixed with 6 times the volume of deionized water.

[0028] The specific plan is as follows:

[0029] A biological fermentation preparation method of quercetin is carried out according to the following steps:

[0030] Step 1, according to the formula ratio and the manufacturing amount, select appropriate amounts of Sophora japonica seed (or rutin), corn flour, bran, glucose, fructose, glycerol, urea, acid-hydrolyzed soybean protein, KH2PO4, MgSO4, ZnSO4, VB2, and VC; the main components of the 1L culture medium used include Sophora japonica seed 20-40g (or rutin 5-10g), corn flour 5-30g, bran 5-55g, glucose 10-50g, fructose 1-40g, glycerol 5-45g, urea 1-4g, acid-hydrolyzed soybean protein 1-4g, KH2PO4 1-2.4g, MgSO4 0.5-2.0g, ZnSO4 0.01-0.05g, VB2 0.01-0.04g, and VC 0.01-0.06g.

[0031] Step 2, dissolve the above-mentioned Sophora japonica seeds (or rutin), corn flour, bran, glucose, fructose, glycerol, urea, acid-hydrolyzed soy protein, KH2PO4, MgSO4, ZnSO4, VB2, and VC in an appropriate amount of water, put into a fermentation tank and sterilize at 121°C for 30 minutes;

[0032] Step 3, using the preserved test tube strain of Coprinus comatus as the object, inoculating the Coprinus comatus strain into a potato glucose medium, culturing at 20-32° C. for 3-7 days, and preserving at 2-10° C. to obtain a Coprinus comatus slant strain;

[0033] Step 4, the Coprinus comatus slant spawn obtained in step 3 is cut into 3mm × 3mm size, 4-6 pieces are picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 100-200rpm, 20-32°C for 3-7 days to obtain a liquid shake flask spawn; wherein each 1L liquid shake flask medium is composed of: corn flour 5-15g, bran 5-25g, grape 5-25g, fructose 1-20g, glycerol 2-20g, acid hydrolyzed soy protein 0.5-2g, KH2PO4 1-2.4g, MgSO4 0.5-2.0g, ZnSO4 0.01-0.05g, VB2 0.01-0.04g, VC 0.01-0.06g;

[0034] Step 5, inoculate the liquid shake flask strain obtained in step 4 into the seed tank culture medium at an inoculation rate of 5-20% (ratio of shake flask strain volume to culture medium volume), culture at 20-32°C, ventilation volume 0.1-1.2 / min (v / v / min, i.e. ventilation volume / liquid volume in the seed tank / min), rotation speed 60-150 rpm for 2-4 days to obtain the seed tank liquid strain; wherein each 1L seed tank culture medium contains: 2-10g of Sophora japonica seed (or 1-5g of rutin), 5-15g of corn flour, 5-25g of bran, 5-25g of glucose, 1-20g of fructose, 2-20g of glycerol, 0.5-2g of acid hydrolyzed soy protein, 1-2.4g of KH2PO4, 0.5-2.0g of MgSO4, 0.01-0.05g of ZnSO4, 0.01-0.04g of VB2, 0.01-0.06gg;

[0035] Step 6: Using liquid fermentation technology, the seed tank bacteria are aseptically transferred to the fermentation medium at an inoculation rate of 5-20% (seed bacteria volume / fermentation medium volume), and cultured at 20-32° C., aeration volume of 0.1-1.2 (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 60-150 rpm for 24-144 h to obtain a Coprinus comatus fermentation liquid containing quercetin.

[0036] Step 7: centrifuge the fermentation liquid in step 6 at 4000 rpm for 10 min, dry the residue at 80° C., and obtain Coprinus comatus mycelium containing quercetin.

[0037] The method for separating and purifying quercetin from Coprinus comatus mycelium is carried out according to the following steps:

[0038] (a) mixing the quercetin-containing mycelium of Coprinus comatus obtained in step 7 with 30 times the volume of anhydrous ethanol, extracting under reflux at 85° C. for 4 h, and collecting the extract;

[0039] (b) The extract was centrifuged at 5000 r / min for 10 min, and the supernatant was collected; the supernatant was concentrated under vacuum to 1 / 5 of the original volume;

[0040] (c) The concentrate was mixed with 6 volumes of deionized water, precipitated for 18 h, filtered, and the residue (quercetin) was washed 3 times with 2 volumes of deionized water. The quercetin was dried at 80 °C.

[0041] (d) The dried product was dissolved in 2 volumes of acetone and crystallized at -20°C to obtain pure quercetin;

[0042] Advantages of the present invention: The method of the present invention is used to prepare quercetin, which not only has a high rutin conversion rate (>95%), a high extraction rate (>90%), and a high quercetin purity (>98%), but also maintains the original conformation and structure of natural quercetin, has no by-products, and has a low impurity content. At the same time, it improves resource utilization and reduces environmental pollution caused by the extraction of a large amount of plant raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1: HPLC chromatograms of standard (A), mycelial extract (B) and blank (C)

[0044] Figure 2: MRM profile of HPLC-MS / MS analysis of standards

[0045] Figure 3: MRM profile of HPLC-MS / MS analysis of mycelial extracts. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with specific embodiments.

[0047] Example 1 Preparation of Coprinus comatus mycelium containing quercetin

[0048] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 20°C for 7 days, and stored at 10°C to obtain a Coprinus comatus slant strain;

[0049] b. The Coprinus comatus slant obtained in step a was cut into 3mm×3mm size, 4 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 200rpm and 32°C for 3 days to obtain a liquid shake flask culture; wherein each 1L liquid shake flask culture medium was composed of: 5g corn flour, 5g bran, 5g glucose, 20g fructose, 10g glycerol, 2g acid hydrolyzed soy protein, 2.4g KH2PO4, 2.0g MgSO4, 0.05g ZnSO4, 0.02g VB2, 0.01g VC;

[0050] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 5% (ratio of shake flask strain volume to medium volume), at 20°C, aeration volume 0.1 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), speed 60 rpm for 2 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: 2g Sophora japonica rice, 5g corn flour, 5g bran, 5g glucose, 20g fructose, 10g glycerol, 2g acid hydrolyzed soy protein, 2.4g KH2PO4, 2.0g MgSO4, 0.05g ZnSO4, 0.02g VB2, 0.01g VC;

[0051] d. Liquid fermentation culture: The inoculum in the seed tank was aseptically transferred to the fermentation medium at an inoculum volume of 5% (seed volume / fermentation medium volume), and cultured for 144 hours at 20°C, aeration volume of 0.1 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 60 rpm to obtain a fermentation liquid of Coprinus comatus containing quercetin; the main components of the 1L culture medium used included 20g of Sophora japonica seed, 30g of corn flour, 5g of bran, 50g of glucose, 40g of fructose, 45g of glycerol, 4g of urea, 1g of acid-hydrolyzed soy protein, 2.4g of KH2PO4, 0.5g of MgSO4, 0.03g of ZnSO4, 0.03g of VB2, and 0.06g of VC;

[0052] e. The fermentation broth in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80°C to obtain Coprinus comatus mycelium containing quercetin.

[0053] Example 2 Preparation of Coprinus comatus mycelium containing quercetin

[0054] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 32°C for 3 days, and stored at 2°C to obtain a Coprinus comatus slant strain;

[0055] b. The Coprinus comatus slant obtained in step a was cut into 3mm×3mm size, 6 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 100rpm and 26°C for 7 days to obtain a liquid shake flask culture; wherein each 1L of liquid shake flask culture medium was composed of: 15g corn flour, 25g bran, 15g glucose, 1g fructose, 10g glycerol, 0.5g acid hydrolyzed soy protein, 1.3g KH2PO4, 1.2g MgSO4, 0.03g ZnSO4, 0.02g VB2, 0.01g VC;

[0056] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 10% (ratio of shake flask strain volume to medium volume), and cultured at 28°C, aeration volume of 0.8 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), and speed of 90 rpm for 4 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: 6g of Sophora japonica rice, 15g of corn flour, 25g of bran, 15g of glucose, 1g of fructose, 10g of glycerol, 0.5g of acid-hydrolyzed soy protein, 1.3g of KH2PO4, 1.2g of MgSO4, 0.03g of ZnSO4, 0.02g of VB2, and 0.01g of VC;

[0057] d. Liquid fermentation culture: The inoculum in the seed tank was aseptically transferred to the fermentation medium at an inoculum volume of 10% (seed volume / fermentation medium volume), and cultured at 28°C, aeration volume of 0.8 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 90 rpm for 60 h to obtain a fermentation liquid of Coprinus comatus containing quercetin; the main components of the 1L culture medium used included 30 g of Sophora japonica seed, 5 g of corn flour, 5 g of bran, 30 g of glucose, 1 g of fructose, 5 g of glycerol, 2.5 g of urea, 2.5 g of acid-hydrolyzed soy protein, 1.7 g of KH2PO4, 2.0 g of MgSO4, 0.03 g of ZnSO4, 0.04 g of VB2, and 0.03 g of VC;

[0058] e. The fermentation broth in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80°C to obtain Coprinus comatus mycelium containing quercetin.

[0059] Example 3 Preparation of Coprinus comatus mycelium containing quercetin

[0060] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 24°C for 4 days, and stored at 8°C to obtain a Coprinus comatus slant strain;

[0061] b. The Coprinus comatus slant obtained in step a was cut into 3mm × 3mm size, 5 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 200rpm and 20°C for 5 days to obtain a liquid shake flask culture; wherein each 1L liquid shake flask culture medium was composed of: corn flour 15g, bran 15g, glucose 15g, fructose 20g, glycerol 2g, acid hydrolyzed soy protein 1.2g, KH2PO4 1g, MgSO4 1.2g, ZnSO4 0.05g, VB2 0.04g, VC 0.04g;

[0062] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 15% (ratio of shake flask strain volume to medium volume), and cultured at 32°C, aeration volume 1.2 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), and speed 60 rpm for 2 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: Sophora japonica 8g, corn flour 15g, bran 15g, glucose 15g, fructose 20g, glycerol 2g, acid hydrolyzed soy protein 1.2g, KH2PO4 1g, MgSO4 1.2g, ZnSO4 0.05g, VB2 0.04g, VC 0.04g;

[0063] d. Liquid fermentation culture: The inoculum in the seed tank was aseptically transferred to the fermentation medium at an inoculation rate of 15% (seed volume / fermentation medium volume), and cultured at 32°C, aeration volume of 1.2 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 60 rpm for 24 h to obtain a fermentation liquid of Coprinus comatus containing quercetin; the main components of the 1L culture medium used included 30g of Sophora japonica seed, 17g of corn flour, 30g of bran, 10g of glucose, 20g of fructose, 25g of glycerol, 4g of urea, 4g of acid-hydrolyzed soy protein, 1.7g of KH2PO4, 1.3g of MgSO4, 0.05g of ZnSO4, 0.03g of VB2, and 0.01g of VC;

[0064] e. The fermentation broth in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80°C to obtain Coprinus comatus mycelium containing quercetin.

[0065] Example 4 Preparation of Coprinus comatus mycelium containing quercetin

[0066] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 28°C for 5 days, and stored at 6°C to obtain a Coprinus comatus slant strain;

[0067] b. The Coprinus comatus slant obtained in step a was cut into 3mm×3mm size, 5 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 150rpm and 32°C for 5 days to obtain a liquid shake flask culture; wherein each 1L liquid shake flask culture medium was composed of: 10g corn flour, 15g bran, 25g glucose, 11g fructose, 20g glycerol, 1.2g acid hydrolyzed soy protein, 1g KH2PO4, 0.5g MgSO4, 0.01g ZnSO4, 0.01g VB2, 0.06g VC;

[0068] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 20% (ratio of shake flask strain volume to medium volume), and cultured at 24°C, aeration volume of 0.4 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), and a speed of 120 rpm for 3 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: 10g of Sophora japonica rice, 10g of corn flour, 15g of bran, 25g of glucose, 11g of fructose, 20g of glycerol, 1.2g of acid-hydrolyzed soy protein, 1g of KH2PO4, 0.5g of MgSO4, 0.01g of ZnSO4, 0.01g of VB2, and 0.06g of VC;

[0069] d. Liquid fermentation culture: The inoculation amount of the seed tank strain was aseptically transferred to the fermentation medium at 20% (seed strain volume / fermentation medium volume), and the culture was carried out at 24°C, aeration volume of 0.4 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 120 rpm for 96 h to obtain the Coprinus comatus fermentation liquid containing quercetin; the main components of the 1L culture medium used included 40g of Sophora japonica seed, 17g of corn flour, 55g of bran, 50g of glucose, 40g of fructose, 5g of glycerol, 1g of urea, 2.5g of acid-hydrolyzed soy egg, 1.2g of KH2PO4, 1.3g of MgSO4, 0.01g of ZnSO4, 0.01g of VB2, and 0.06g of VC;

[0070] e. The fermentation broth in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80°C to obtain Coprinus comatus mycelium containing quercetin.

[0071] Example 5 Preparation of Coprinus comatus mycelium containing quercetin

[0072] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 20°C for 3 days, and stored at 2°C to obtain a Coprinus comatus slant strain;

[0073] b. The Coprinus comatus slant obtained in step a was cut into 3mm×3mm size, 4 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 200rpm and 26°C for 5 days to obtain a liquid shake flask culture; wherein each 1L liquid shake flask culture medium was composed of: 5g corn flour, 25g bran, 25g glucose, 11g fructose, 10g glycerol, 2g acid hydrolyzed soy protein, 1g KH2PO4, 2.0g MgSO4, 0.03g ZnSO4, 0.04g VB2, 0.06g VC;

[0074] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 20% (ratio of shake flask strain volume to medium volume), and cultured at 24°C, aeration volume of 1.2 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), and a speed of 120 rpm for 4 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: 5g rutin, 5g corn flour, 25g bran, 25g glucose, 11g fructose, 10g glycerol, 2g acid hydrolyzed soy protein, 1g KH2PO4, 2.0g MgSO4, 0.03g ZnSO4, 0.04g VB2, 0.06g VC;

[0075] d. Liquid fermentation culture: The inoculation amount of the seed tank strain was aseptically transferred to the fermentation medium at 20% (seed strain volume / fermentation medium volume), and the culture was carried out at 32°C, aeration volume of 1.2 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 120 rpm for 144 h to obtain a Coprinus comatus fermentation liquid containing quercetin; the main components of the 1L culture medium used included 10g rutin, 30g corn flour, 55g bran, 10g glucose, 20g fructose, 25g glycerol, 2.5g urea, 2.5g acid hydrolyzed soy protein, 2.4g KH2PO4, 0.5g MgSO4, 0.03g ZnSO4, 0.04g VB2, and 0.01g VC;

[0076] e. The fermentation liquid in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80° C. to obtain Coprinus comatus mycelium containing quercetin.

[0077] Example 6 Preparation of Coprinus comatus mycelium containing quercetin

[0078] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 26°C for 5 days, and stored at 6°C to obtain a Coprinus comatus slant strain;

[0079] b. The Coprinus comatus slant obtained in step a was cut into 3mm×3mm size, 5 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 150rpm and 26°C for 3 days to obtain a liquid shake flask culture; wherein each 1L liquid shake flask culture medium was composed of: 15g corn flour, 10g bran, 15g glucose, 20g fructose, 2g glycerol, 0.5g acid hydrolyzed soy protein, 2.4g KH2PO4, 1.2g MgSO4, 0.01g ZnSO4, 0.02g VB2, 0.04g VC;

[0080] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 12% (ratio of shake flask strain volume to medium volume), and cultured at 32°C, aeration volume of 0.8 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), and a speed of 90 rpm for 2 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: 3g rutin, 15g corn flour, 10g bran, 15g glucose, 20g fructose, 2g glycerol, 0.5g acid hydrolyzed soy protein, 2.4g KH2PO4, 1.2g MgSO4, 0.01g ZnSO4, 0.02g VB2, and 0.04g VC;

[0081] d. Liquid fermentation culture: The seed tank strain was aseptically transferred to the fermentation medium at an inoculation rate of 12% (seed strain volume / fermentation medium volume), and cultured at 32°C, aeration volume of 0.8 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 90 rpm for 68 h to obtain a Coprinus comatus fermentation liquid containing quercetin; the main components of the 1L culture medium used included 7.5 g rutin, 17 g corn flour, 5 g bran, 50 g glucose, 40 g fructose, 45 g glycerol, 1 g urea, 4 g acid-hydrolyzed soy protein, 1 g KH2PO4, 2.0 g MgSO4, 0.05 g ZnSO4, 0.01 g VB2, and 0.03 g VC;

[0082] e. The fermentation liquid in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80° C. to obtain Coprinus comatus mycelium containing quercetin.

[0083] Example 7 Preparation of Coprinus comatus mycelium containing quercetin

[0084] a. Using a preserved test tube strain of Coprinus comatus (strain number UJS-1006, obtained from Nantong Anhui Biotechnology Co., Ltd.) as the object, the Coprinus comatus strain was inoculated into a potato glucose medium, cultured at 28°C for 7 days, and stored at 10°C to obtain a Coprinus comatus slant strain;

[0085] b. The Coprinus comatus slant obtained in step a was cut into 3mm×3mm size, 6 pieces were picked and inoculated into a shake flask containing a liquid shake flask medium, and cultured in a shaker at 100rpm and 20°C for 7 days to obtain a liquid shake flask culture; wherein each 1L of liquid shake flask culture medium was composed of: 10g corn flour, 5g bran, 5g glucose, 1g fructose, 20g glycerol, 1.2g acid hydrolyzed soy protein, 1.3g KH2PO4, 0.5g MgSO4, 0.05g ZnSO4, 0.01g VB2, 0.01g VC;

[0086] c. The liquid shake flask strain obtained in step b was inoculated into the seed tank medium at an inoculation rate of 5% (ratio of shake flask strain volume to medium volume), and cultured at 28°C, aeration volume of 0.4 / min (v / v / min, i.e., aeration volume / liquid volume in the seed tank / min), and a speed of 150 rpm for 3 days to obtain a seed tank liquid strain; wherein each 1L seed tank medium contains: 1g rutin, 10g corn flour, 5g bran, 5g glucose, 1g fructose, 20g glycerol, 1.2g acid hydrolyzed soy protein, 1.3g KH2PO4, 0.5g MgSO4, 0.05g ZnSO4, 0.01g VB2, 0.01g VC;

[0087] d. Liquid fermentation culture: The inoculation amount of the seed tank strain was aseptically transferred to the fermentation medium at 5% (seed strain volume / fermentation medium volume), and the culture was carried out at 28°C, aeration volume of 0.4 / min (v / v / min, i.e., aeration volume / liquid volume in the fermentation tank / min), and a stirring speed of 150 rpm for 96 h to obtain a Coprinus comatus fermentation liquid containing quercetin; the main components of the 1L culture medium used included 5g rutin, 5g corn flour, 30g bran, 30g glucose, 40g fructose, 5g glycerol, 4g urea, 1g acid-hydrolyzed soy protein, 1.2g KH2PO4, 1.3g MgSO4, 0.01g ZnSO4, 0.03g VB2, and 0.06g VC;

[0088] e. The fermentation broth in step d was centrifuged at 4000 rpm for 10 min, and the residue was dried at 80°C to obtain Coprinus comatus mycelium containing quercetin.

[0089] Example 8 Extraction of Quercetin

[0090] (a) mixing the quercetin-containing mycelium of Coprinus comatus obtained in the above example with 30 times the volume of anhydrous ethanol, extracting under reflux at 85° C. for 4 h, and collecting the extract;

[0091] (b) The extract was centrifuged at 5000 r / min for 10 min, and the supernatant was collected; the supernatant was concentrated under vacuum to 1 / 5 of the original volume;

[0092] (c) The concentrate was mixed with 6 volumes of deionized water, precipitated for 18 h, filtered, and the residue (quercetin) was washed 3 times with 2 volumes of deionized water. The quercetin was dried at 80 °C.

[0093] (d) The dried product was dissolved in 2 volumes of acetone and crystallized at -20°C to obtain pure quercetin;

[0094] (e) The weight of quercetin isolated from 1 L of mycelium in the fermentation broth of Examples 1-7 using this extraction method is shown in Table 1.

[0095] Table 1. Weight of quercetin in 1L fermentation broth mycelium of Examples 1-7

[0096]

[0097] Example 9: Determination of quercetin content by high performance liquid chromatography

[0098] (a) Sample preparation: Weigh 1 mg of quercetin sample, dissolve it in 50 mL of ethanol, centrifuge at 5000 r / min for 10 min, filter the supernatant with a syringe filter, and use the filtrate for determination.

[0099] (b) Preparation of standard curve: Accurately weigh 5 mg of quercetin standard solution dried to constant weight at 105℃ into a 50mL beaker, add a small amount of anhydrous ethanol to dissolve, then pour into a 50mL volumetric flask to make up to the mark, shake well and set aside. Pipette 0, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mL of quercetin standard solution into a 10mL brown volumetric flask, make up to the mark with anhydrous ethanol, and inject 1mL of each solution. Use quercetin concentration as the ordinate and absorbance as the abscissa to draw a standard curve for quercetin and establish a regression equation.

[0100] (c) 1 mL of the pretreated sample filtrate was injected, the peak area was determined, and the quercetin content was calculated according to the standard curve regression equation.

[0101] (d) The purity of quercetin in samples of Examples 1-7 was measured by colorimetry and is shown in Table 2.

[0102] Table 2: Purity of quercetin in fermentation broth of Examples 1-7 (%)

[0103]

[0104] Although the present invention has been illustrated and described with respect to the preferred embodiments, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope of the present invention as defined by the claims.

Claims

1. A method for preparing quercetin by biological fermentation, characterized in that: The steps include: Step 1, inoculating the Coprinus comatus strain into a potato glucose medium to obtain a Coprinus comatus slant strain; Step 2, inoculating the Coprinus comatus slant strain obtained in step 1 into a shake flask culture medium to obtain a shake flask strain; Step 3, inoculating the shake flask strain obtained in step 2 into the seed tank culture medium at a volume ratio of 5-20%, and cultivating to obtain the seed tank strain; Step 4, aseptically transferring the seed tank strain obtained in step 3 into a fermentation medium at an inoculation rate of 5-20% by volume, and culturing to obtain a fermentation liquid containing quercetin; Step 5, separating and purifying the fermentation broth obtained in step 4 to obtain pure quercetin; Each 1L shake flask medium contains: 5g corn flour, 25g bran, 25g glucose, 11g fructose, 10g glycerol, 2g acid hydrolyzed soy protein, 1g KH2PO4, 2.0g MgSO4, 0.03g ZnSO4, 0.04g VB2, 0.06g VC; Each 1L seed tank culture medium contains: 5g rutin, 5g corn flour, 25g bran, 25g glucose, 11g fructose, 10g glycerol, 2g acid hydrolyzed soy protein, 1g KH2PO4, 2.0g MgSO4, 0.03g ZnSO4, 0.04g VB2, 0.06g VC; Each 1L of fermentation medium contains: 10g rutin, 30g corn flour, 55g bran, 10g glucose, 20g fructose, 25g glycerol, 2.5g urea, 2.5g acid-hydrolyzed soy protein, 2.4g KH2PO4, 0.5g MgSO4, 0.03g ZnSO4, 0.04g VB2, and 0.01g VC.

2. The preparation method according to claim 1, characterized in that: The culture conditions of step 2 are 100-200 rpm, 20-32° C., and culture for 3-7 days.

3. The preparation method according to claim 2, characterized in that: The culture conditions of step 2 are 100-200 rpm, 20-26° C., and culture for 5-7 days; or 100-200 rpm, 26-32° C., and culture for 3-5 days.

4. The preparation method according to claim 1, characterized in that: The culture conditions of step 3 are 20-32° C., ventilation volume 0.1-1.2 v / v / min, rotation speed 60-150 rpm for 2-4 days.

5. The preparation method according to claim 4, characterized in that: The culture conditions of step 3 are 24-32° C., ventilation volume 0.4-1.2 v / v / min, rotation speed 60-120 rpm for 2-4 days.

6. The preparation method according to claim 1, characterized in that: The culture conditions of step 4 are 20-32° C., aeration volume 0.1-1.2 v / v / min, rotation speed 60-150 rpm for 24-144 hours.

7. The preparation method according to claim 6, characterized in that: The culture conditions of step 4 are 24-32° C., aeration volume 0.4-1.2 v / v / min, rotation speed 60-150 rpm for 68-144 hours.

8. The preparation method according to claim 1, characterized in that: The separation and purification in step 5 further comprises the following steps: Step a, centrifuging the fermentation liquid and drying the residue to obtain Coprinus comatus mycelium containing quercetin; Step b, extracting the mycelium of Coprinus comatus by reflux extraction with anhydrous ethanol, concentrating the extract and mixing it with deionized water, precipitating, filtering, washing the filter residue with deionized water, and drying; Step c: dissolving the dried product with acetone and crystallizing it to obtain pure quercetin.

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